Bacteria of the genus Acinetobacter are known to be involved in the degradation, leaching and removal of various hazardous compounds from the environment. Several studies of Acinetobacter spp. have reported on the genes involved in alkane degradation; but less is known about the proteins that are expressed at certain points within the degradation period. Acinetobacter sp. LT1A and Acinetobacter sp. V2 were isolated from diesel- and used engine oil-contaminated soils respectively. In a previous investigation (Toolsi, 2008), these isolates have been shown to demonstrate different gene expression patterns during diesel degradation using real time PCR. The real time PCR data showed that isolate V2 made use of multiple alkane hydroxylases whereas LT1A made use of only one, and the expression of the alkane hydroxylase regulator alkR and secretory protein xcpR also revealed multiple product formations in isolate V2 as compared to LT1A. Thus the objectives for the current investigation were to monitor the hydrocarbon degradation ability of Acinetobacter sp. isolates V2 and LT1A using medium chain (C14) and long chain (C28) hydrocarbon substrates and to compare the hydrocarbon degradation abilities and protein expression patterns of both isolates. To achieve this, the isolates were grown for 20 days in Bushnell Haas liquid medium supplemented with tetradecane (C14) or octocosane (C28) as a sole carbon source. Gravimetric analysis was used to monitor degradation and whole cell protein was extracted from the culture medium throughout the 20-day study period. The protein expression patterns were visualized using 1D and 2D PAGE. The 2D PAGE images were analyzed using the PDQuest Advanced 2D image analysis software (BIORAD). By day 20, approximately 90% of C14 was degraded by both isolates, whereas only 36% of C28 had been broken down. In both the C14 and C28 degradation assays, the isolates achieved significant amounts of hydrocarbon degradation as compared to the abiotic controls. One-dimensional and 2D SDS-PAGE gels indicated that there are observable differences in protein expression patterns between the isolates during C14 and C28 degradation. Both isolates achieved similar rates of hydrocarbon usage, but appear to do so using different, unidentified, protein systems. Analysis of the 2D-SDS PAGE gel images revealed that more proteins were required for the utilization of the long chain alkane (C28) as compared to the medium chain alkane (C14) for both isolates. Potential spots of interest were identified from the 2D SDS-PAGE images and sequenced. The identities of these proteins were found to be: a conserved hypothetical protein, TonB-dependent receptor protein, Peptidyl-prolyl-cis-trans isomerase and a Protein containing DUF1559. No alkane hydroxylase components were detected in this study. This investigation demonstrated the need for more studies at the proteomic level. Future investigations should focus on the insoluble subproteome of the isolates and make use of larger sample sizes (replicates) to reduce variation in spot detection and quantification. Genomic sequencing of the isolates will also shed light on the genetics and biochemistry of alkane metabolism in these Acinetobacter sp. isolates. / Thesis (M.Sc.)-University of KwaZulu-Natal, Westville, 2012.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:ukzn/oai:http://researchspace.ukzn.ac.za:10413/8345 |
| Date | January 2012 |
| Creators | Pretorius, Karyn. |
| Contributors | Lin, Johnson. |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | English |
| Type | Thesis |
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